The project was undertaken to study the latency of JC virus (JCV) in human central nervous system and the possible role of the virus in neurological diseases of unknown origin. In collaboration with the laboratory of Dr. R.J. Frisque, sections of brains and kidneys were screened for the presence of JCV DNA by polymerase chain reaction (PCR). Using regulatory and coding region-specific oligonucleotide primers, more than 5O% of the normal (non-progressive multifocal leukoencephalopathy [PML])brains were found to contain low levels of JCV DNA. Cloning and sequences analysis of the PCR products confirmed the PCR findings. While the archetype strain of JCV was identified in normal kidney samples, PML kidneys and normal brains contained JCV strains resembling strains isolated from PML brain. These findings suggest JCV latency in the normal human brains. The latent virus might persist in the brain without causing demyelinating disease. The reactivation might take place after interaction with an immunocompromising agent, e.g. HIV or due to prolonged immunodeficiency, resulting in PML. The presence of the DNA sequence of the Mad- 1 strain of JCV in the brain tissues from patients with multiple sclerosis (MS) and medulloblastoma reported in the last year's annual report was found later on not to represent true JCV sequences but contamination of the tissues with pBR322 cloned JCV Mad-1 plasmid DNA from ENZO probe (used for in situ hybridization). A tri-primer PCR procedure was developed for the detection of the simultaneous presence of cloned and free viral DNA in tissue samples. The procedure was successfully employed to detect the contaminated cloned DNA in the tissues. During amplification by PCR of the regulatory region of Mad-1 strain of JCV, we found that certain primer pairs can give rise to an artifactual deletion of one copy of a tandem repeat contained within the amplified region. The data indicated that the tandem repeats can be subject to deletion artifacts if one primer is close to the repeat unit and/or the primer binding site encompasses a potential secondary structure. In the case of repeated viral promoter/enhancer elements, these spurious products should not be confused with "archetype" sequences which naturally lack duplication.